Antibiotic w847 (megalomicin)and methods for production thereof

ABSTRACT

A NEW ANTIBIOTIC COMPLEX IDENTIFIED AS ANTIBIOTIC W847 COMPLEX, (OR ANTIBIOTIC W847 OR MAGALOMICIN), COMPOSED OF AT LEAST FOUR MAJOR ANTIBIOTIC COMPONENTS IS DUCED BY THE CULTIVATION UNDER SUBMERGED AEROBIC CONDITIONS OF MICROMONOSPORA MAGALOMICEA, A HITHERTO UNDESCRIBED SPECIES OF THE GENUS MICROMONOSPORA OF THE ORDER ACTINOMYCETALES. THE INDIVIDUAL COMPONENTS ARE ISOLATED BY SOLVENT EXTRACTION AND CHROMATOGRAPHY AND ARE CHARACETERIZED BY THEIR RESPECTIVE CHEMICAL AND BIOLOGOICAL PROPERTIES.

United States Patent Patented June. 25, 1974 ANTIBIOTIC W847 (MEGALOMICIN) AND METHODS FOR PRODUCTION THEREOF Marvin J. Weinstein, East Brunswick, George M. Luedemann, Glen Ridge, Gerald H. Wagman, East Brunswick, and Joseph A. Marquez, Montclair, N.J., assignors to Schering Corporation, Bloomfield, NJ. No Drawing. Application Sept. 4, 1969, Ser. No. 855,424,

now Patent No. 3,632,750, which is a continuation-inpart of application Ser. No. 707,100, Feb. 21, 1968, which in turn is a continuation-impart of application Ser. No. 641,522, May 26, 1967, both now abandoned. Divided and this application June 25, 1971, Ser. No.

Int. Cl. C07c 129/18 US. Cl. 260-210 20 Claims ABSTRACT OF THE DISCLOSURE A new antibiotic complex identified as Antibiotic W847 Complex, (or Antibiotic W847 or megalomicin), composed of at least four major antibiotic components is produced by the cultivation under submerged aerobic conditions of Micromonospara megalomicea, a hitherto undescribed species of the genus Micromonospora of the order Actinomycetales. The indivdual components are isolated by solvent extraction and chromatography and are characterized by their respective chemical and biologoical properties.

FIELD OF THE INVENTION DESCRIPTION OF THE INVENTION The Microorganism The hitherto undescribed species of the genus Micromonospora employed in the production of the new antibiotics of this invention was isolated originally from a soil sample obtained directly from the earth by one of the inventors named herein. Two strains of this M icromonospora species have been isolated. These two strains are natural color variants and may be distinguished microscopically by the degree to which sporulation occurs. Cultures of living organisms of these two strains have been deposited and made a part of the stock culture collection of the United StatesDepartment of Agriculture, Northern Utilization Research and Development Division, Peoria, Illinois, where they have been designated N'R RL 3274 and NRRL 3275. These strains are available from the collection agency upon request. These strains will be referred to hereinafter individually as Micromonospora sp. W847 :(NRRL 3274) and as Micromonospora sp. W847 (NRRL 3275) and collectively as Micromonospora sp.

W847. These strains are also referred to by applicants as Micromonospora.megalomicea var megalomicea NRRL 3274 and M ic romonospora megalomicea var nigra NRR-L 3275, respectively. These names are deemed to be synony-' mous with those set forth above and may be used interchangeably. I

Macroscopic examination of 28 day old cultures (24- 26" C.) of Micromonospora sp. W847 (NRRL 3274) on Czapeks Sucrose Agar is characterized by good growth exhibitingno aerial mycelium and by raised and plicate; colonies of a soft, moist and non-waxyconsistency pro ducing no diflfusablc pigment. The color of the colonysurface, described according to the Descriptive Color Name Dictionary, Taylor, Knoche and Granville, published by the Container Corporation of America, 1950 (USA), together with the color chip number taken from the Color Harmony Manna 4th Edition, 1958, published by-thesame company, is orange m4-la. This colorcorrespond-s to the synonym or near synonym, strong orange 50, as found in the National Bureau of Standards Circular 'No.-; 553, Nov. 1,. 1955, USA (all colony color designationsemployed hereafter are based upon the same color refer ence system).

Microscopic examination of the same culture shows aregular mycelium composed of long branched filaments approximately 0.6 in diameter. Spores are-infrequently found.

Macroscopic examination of 28 day old cultures of Micromonospora sp. W847 (NRRL 3275), grown on the same medium and under the same conditions described above, is characterized by good growth exhibiting the same gross colony characteristics as the previous strain except for color. The color of the colony surface is black. Microscopically, this culture shows a regular mycelium composed of long branched filaments approximately 036 in diameter. In this strain, however, spores are very abundant and are randomly produced throughout 'the mycelium. The spores are ovoid to spherical in shape with a O.71.0,u diameter and are dark brown when mature.

Colonies of M icromonospora sp. W847 may be further characterized by their growth characteristics on various nutrient media. Table 1 below gives the growth char-- acterist-ics of Micromonospora sp. W847 on some of the 1 more widely used nutrient media. The colony observations were made on 14 day old (24-26 C.) cultures unless;

otherwise specified.

TABLE 1 Colony characteristics of Micromo'nospora sp. W847 on various media Glucose asparag'ine ag Growth poor. I Aerobic or anaerobic. Aerobic.

Bennett's aga.r Grovlgth poor.

Emerson's agar Tomato paste oatmeal agar Do.

Glucose-yeast extract agar Gronwto good, raised, finely p ea e.

Potato slice wth poor.

Gro Sucros)e nitrate agar (Czapek's Growth good, raised, plicate.

agar

- Tyrosine agar, observations at 2, 7, Growth fair, crystals poorly dlsand 14 days (After Gordon and solved directly under colony, Smith J. Bact. 69:147).

brown difiusible, pigment produced alter 3 weeks, difl'usible pigment not black or typical of a tyrosine positive as produced by streptomycos.

Micromonospora sp. W847 shows good growth at temperatures between 26 and 37 C. and no growth at 50 C. It is aerobic and does not reduce nitrate to nitrite. This .species does-not liquify gelatin; does not hydrolyze milk and does not decompose cellulose. No difiusible pigment is produced on peptone-iron agar.

Micromonospora sp. W847 shows good growth on carv bohydrate media containing 0.5 percent yeast extract base .and 1 percent of l-arabinose, glucose, starch, and sucrose but only fair growth where the carbohydrate source is 1 percent xylose. It grows poorly, if at all, on such media in which the solo carbohydrate source is 1 percent d-arabinose, cellulose, dulcitol, galactose, glycerol, lactose, levulose, melibiose, melisitose, rafiinose, rhamnose, ribose,

inositol or mannitol.

. TABLE 2.UTILIZATION= OF NITROGEN-SOURCES Nitrogen source plus 17 glucose Growth characteristics 0.5% Dil'eo yeast extract Growth good, raided, finely plicate. 1.0% NZ amine type.A"-.... Growth fair. 1% asparagine Growth poor. 1% 'glutamic acid Do 1% ammonium nitrate Product of Sheifield Chemical Company, Norwich, N.Y.

The antibiotic hereinafter to be described is prepared through utilization of Micromonospora sp... W847 var. ZYRRL 3274 and var. NRRL 3275. The instant invention however, is not to be construed as so limited as to exclude the use of other variants of Micromonospora sp. W847 or mutants thereof produced from this organism by mutating agents such as, for example, high frequency radiation including X-ray and ultraviolet, actinophages and nitrogenmustard. In the following description wherein the process for antibiotic production is elaborated, it is to be understood that the use of the term, Micromonospora sp. W847, is intended to include the color variants specifically describedabove as well 'as all other fully equivalent variants and mutants of'this species.

. THE ANTIBIOTIC (1) Preparation of Antibiotic W847 Complex Micromonospora sp. W847, by controlled fermentation techniques as described hereafter, produces a complex mixture of antibiotic substances hereinafter referred to as the Antibiotic W847 Complex. This antibiotic complex may also be referred to as Antibiotic W847 or megalomicin; the names are synonymous and may be used interchangeably.

In order to prepare a suitable inoculum for subsequent fermentation, Micromonospora sp. W847 is grown under submerged aerobic conditions with continual agitation at temperatures ranging from about 22 to about. 37 C. for a period of from about 2 to about 4 days. Generally, a temperature of 35 C. over a 3 day period is found most satisfactory. The medium normally used in this first germination stage is aqueous and contains quantities of car bon and nitrogen sources assimilable by the organisms. The alkalinity of the media desirably is adjusted to about pH 7.5 before autoclavingby the addition "of base, such as sodium hydroxide. 3

Exemplary of media employed in the germination stage, are the following: Medium 1: Bacto-Beef Extract (Difco), 3.0 gm; Bacto-Tryptone (Difco), 5.0 gm.; dextrose, 1.0 gm.; starch (potato), 24.0 gm.; Bacto-Yeast -Extract (Difco), 5.0 gm.; CaCO 2.0 gm.; tap water,.1000 ml.; Medium 2: Bacto-Yeast Extract (Difco); 5.0 gm. fish solubles, 1.0 gm.; corn steep liquor, 1.0 gm.; dextrose, 20.0 gm., CaCO 1.0 gm.; tap water, 1000 ml.

Micromonospora sp. W847 is yields of Antibiotic W847 Complex are obtained follow ing fermentation for 66 hours at 31 C. with introduction of air at a rate of 4.5 liters per minute and with impeller agitation at 250 r.p.m. Using 14 liter fermenters containing 10 liters of medium, yields ranging from 300 to 800.meg./ml. have been obtained under these conditions.

Media useful in the fermentation stage include, for example, the following: Medium 3: sucrose, 30.0 gm.; dextrose, 2.5 gm.; trypticase, 17.0 gm.; NaNO 3.0 gm.; K HPO 3.5 gm.; MgSO .7H O, 0.5 gm.; KCl, 0.5 gm. FeSO 0.01 gm.; NaCl, 5.0 gm.; tap water, '1000 ml.; Medium 4: Bacto-Yeast Extract (Difco), 5.0 gm.; dextrose, 10.0 gm.; starch, 20.0 gm.; casein hydrolysate, 5.0 gm.; CaCO 4.0 gm.; tap water, 1000 ml.; Medium 5: sucrose, 20.0 gm.; peptone, 5.0 gm.; cotton seed protein hydrolysate, 5.0 gm.; CaCO 4.0 gm.; tap Water, 1000 ml.; Medium 6: sucrose, 30.0 gm.; peptone, 8.0 gm.;

gm.; FeSO 0.01 gm.; tap water, 1000 ml. Medium 7: trypticase, 17 gm.; NaCl, 5.0 gm.; K HPO 2.5 gm.; dextrose, 2.5 gm.; Czapek-Dox broth, 35.0 gm.; tap water, q.s. 1000 ml.

For recovery of Antibiotic W847 Complex from the fermentation medium, the Whole broth is adjusted to about pH 9.5 with base such as, for example, sodium hydroxide. The whole broth then is extracted with about 2 volumes for each volume of broth with a suitable immiscible organic solvent, such as, for example, ethyl acetate, methylene dichloride, butyl acetate, chloroform, butanol and the like. The solvent phase is next concentrated undervacuum to a small volume, approximately 100 fold concentration, and is purified by column chromatography employing, for example, LH 20 Sephadex, an alkylated crossllnked dextran (Pharmacia Fine Chemicals, Inc., Uppsala, Sweden), suspended in about 95 percent aqueous ethanol. The column is eluted with the aqueous ethanol. Column fractions are combined according to their antibacterial activities by agar disc testing against Staphylococcus aureus (ATCC 6538P), concentrated to dryness, dissolved in a small amount of ethyl ether or acetone and added to an excess volume of petroleum ether (b.p. about to about 60C.). The mixture is then transferred to a Dry Ice-acetone bath (approximately C.) and allowed to stand for about 20 minutes after which it is allowed to return to room. temperature. The mother liquor is separated from any oily residue by decanting andis concentrated to dryness. Material produced in this manner is usually found to have potency of about 232 mcg./mg. according to the bio-assay procedure described below.

The assay is a cylinder cup assay using Sarcz'na lutea (ATCC 9341) as the test organism. The conditions of the assay are similar to the cylinder plate assay described 'Des irably, a second germination stage (e.g. inoculum Y stage) is employed prior to fermentation and harvesting of the antibiotic in order to enrich the inoculum. Employing media as described above, fresh medium is inoculated with the germinated culture previously prepared (5% w'./v.) and incubated at about 22 to about 37C. for about 2 to about 4 days. Usually, a temperature of 28 C. over a period of 3 days is found most satisfactory.

In order to produce the Antibiotic W847 Complex, a 5. percent (w./v.) inoculum taken from the inoculum stage is added to aqueous fermentation medium which, prior to 'autoclaving, has been adjusted to about pH 7.1 to 7.8. Air, usually about 3 to about 6 liters per minute, is introduced into the fermentation which, generally,*is conducted at a temperature between about 22 to about 37 C. over a period of about 2 to about 6 days with'continuous agitation. Particularly with reference to Medium 3, described below, it has been found that maximum for erythromycin (Grove and Randall, 1955, Med. Encyclopedia, Inc., N.Y.).'A mcg. of AntibioticW847 Complex activity is the amount of material which produces a zonal response of 20.8-30.5 mm. under the conditions of this assay.

Alternatively, Antibiotic W847 Complex may ,be prepared by-utilizing the following fermentation and isolation techniques: a

Employing Medium 1 for the preparation of an inoculum, fermentation flasks containing this medium are inoculated with a culture (approximately 5% w./v.) of Micromonospora sp. W847. The flasks are placed on a rotary shaker running at about 280 strokes per minute and are incubated at 28 C. for 72 hours. At the end of this time, fermentors, each'charged with fermentation medium (Medium 7 for example), adjusted to pH 7.15 to 7.25, are inoculated withapproximately'5% (v./v.) of the 72' hour inoculum culture. The fermentation medium is agitated at a rate of about 500 r.p.m. at a temperature of 31 C. Air flow is induced through the medium at a rate of about 0.5 liters of air/liter of broth/minute. The agitation rate may be increased to 600 r.p.m. after 24 utes. The agar plate is incubated at 37 C. overnight and the zones of inhibition evaluated.

Results of these experiments are shown in Table 4 below.

TABLE 4.-COMPARATIVE THIN LAYllgMgillggMAToGRAPHY OF ANTIBIOTIC W847 R1 of inhi- System Antibiotic R: and color by H1804 spray zone Chloroform:methanol:17% ammonia Antibiotic W847 complex Blue black, 0.98-.- 0. 98 2:1:1 (v./v.). Oleandomycin Green, 0.98 0.98

' Yellow tan, 0.98... 0.98

Magnamycin Blue purple, 0.98 0. 98

Butanohacetic acid:water 321:1 Antibiotic W847 complex Red purple, 0.13 0.13 (v /v,), Crythromycin Green brown, 0.26 0.26 Spiramycim. Purple, gray, 0.06, 0.06

Oleandomyci Green, 0.19 0. 19

Magnamycin Purple, red purple, 0.40, 0.47 0. 40

The pooled fermentation broth is adjusted to about pH 9.25 with aqueous sodium hydroxide (50%) and is extracted withabout 2 volumes of ethyl acetate and the extracts are concentrated to about one to two percent of the original volume. The ethyl acetate concentrate is then extracted at least twice with half volumes of hydrochloric acid (about 0.14 N). Alternatively, the acid extraction can be carried out with other mineral acids at about the same normality. The combined acid extracts are made slightly alkaline with 5% aqueous sodium hydroxide (to about pH 8.5 to 9.0) and are extracted at least twice with about equal volumes of ethyl acetate. The combined ethyl acetate extracts are concentrated to dryness under reduced pressure to obtain the Antibiotic W847 Complex. The complex at this stage will show a potency of 450 to 550 meg/mg. in the assay described above.

(2 Chemical Characterization of Antibiotic W847 Complex Antibiotic W847 Complex was compared by paper chromatography with a variety of antibiotics, including several macrolides. In a series of solvent systems, as shown in Table. 3 below, Antibiotic W847 Complex moves as a single spotand is diflerentiated from all other antibiotics except the macrolides which were tested (i.e. magnamycin, oleandomycin, spiramycin and erythromycin).

Further, by comparison of the physical, chemical and biological properties of Antibiotic W847 Complex, and of its separated fractions, as hereinafter described with properties of known macrolides as reported in the literature, Antiobotic W847 Complex and its components are distinguishable from all known macrolide antibiotics.

Antibiotic W847 Complex gives a positive color reaction in the Ninhydrin, Starch-KI and Biuret tests and a negative color reaction in the Stannous Chloride, Molisch and Sagaguchi tests. The complex is essentially transparent in the ultraviolet region. The activity of Antibiotic W847 Complex is not significantly altered when a solution thereof is subjected to a temperature of 100 C. for 30 minutes throughout the pH range of 2 to 10 (stability is tested by dissolving the antibiotic in ethanol, diluting with buffer, and disc testing against S. aureus and Escherichia coli) The complex shows no loss in activity after treatment with trypsin, chymotrypsin, pepsin, a-amylase or penicil linase in buffered solution (optimal pH for each enzyme) at 37 C. for up to 24 hours exposure. Antibiotic W847 Complex is soluble in most polar organic solvents such as, for example, ethyl acetate, methanol and acetone.

(3) Separation of Antibiotic W847 Components Bioautography of thin layer silica gel plates on agar seeded with Sarcina lutea following chromatography in a TABLE 3.OOMPARATIVE R: VALUES OF ANTIBIOTIC W847 COMPLEX Rrs of antibiotics Antibiotic W847 Ole- Chlor- Chlor- Paper chromato- Com- Magnaando- Erythro- Spira- Peni- Kanaamphentetra- Genta- Strepto- Tetragraphic systems plex mycin mycin mycin mycin cillin mycln icol cycline micin mycin cycline 80% methanol plus 3% sodium chloride (w./v.) 1:1 descending 0.90 0. 00 0.90 0.90 0.90 0.63 0.30 0.84 0.74- 0.59 0.57 0.72 Propanol pyridine: acetic acid:water (6:4:1:3) ascending (v./v.) 0.93 0.93 0.93 0.93 0.93 0.91 0.18 0.91 0.75 0.26 0.40 0.70 Butanohacetic acid:

water (4:1:5) ascending (v./v.) 0.78 0.78 0.78 0. 78 0. 78 0.85 0.00 0.92 0.46 0.00 0.00 0.38 80% phenol ascending (v./v.) 0.86 0.86 0.86 0.86 0. 86 0.81 0.17 0.93 0.87 0.50 0.00 I i 0.83

Paper buffered with 0.95 molar Na2SO4 0.05 molar NaHSO4.

Diiferentiation of Antibiotic'W847 Complex from these important macrolide antibiotics was made by thin layer chromatograph' on silica gel GF plates (Analtech Inc., Wilmington, Del.). Zones were detected by the following twomethods:

l. Plates are sprayed with a mixture of concentrated sulfuric acid-methanol (1 :1 v./v.) and developed by heating at 105 C. for several minutes. The zones of organic material appear as colored areas'against a white back-' ground. 1

2. Plates are allowed to remain in contact with an agar layerseeded with S. aureus. A sheet'of Whatman No. 1 paper is placedbetween the seeded agar layer and the plate to insure that the silica gel does not adhereto the agar. The paper and silica .gel are removed after 15. minsolvent system consisting of chlorofor'mzmethanol (60:40 v./v.) indicates that Antibiotic W847 Complex consists of at least four antibiotic components which will be identified hereinafter as Antibiotic W847 Fractions A, B, C .and C (or as Antibiotic W847-A, Antibiotic W847-B, Anti biotic W847-C and Antibiotic W847-C These fractions may also be referred to as megalomicin-A, megalomicin-B, magalomicin-C and megalomicin-C respectively. These fractions, in the solvent system described above, show R; values of 0.19 (Fraction A), 0.38 (Fraction B), 0.52 (Fraction C and 0.65 (Fraction C,) respectively.

Separation of the fractions is achieved by dissolving Antibiotic W847 Complex in acetone mixed with silicic acid, evaporating the acetone and adding the dry mixture to the top of a silicic acid column. The column is eluted with chloroform:methanol (60:40 v./v.) and fractions are collected. The column is monitored by disc testing each fraction against S. aureus and E. coli. The active fractions are chromatographed on silica gel thin layer plates run for about 1.5 hours in the same solvent system used for the column. The pattern of the antibiotic fractions are detected by the two methods previously described. Fractions are combined according to their chromatographic patterns and are concentrated.

By this method, Fraction C as a hydrochloride salt is separated and isolated as a white powder by dissolving the pooled active fractions in methanol and precipitating with about volumes of ethyl ether. Fractions C hydrochloride at this stage exhibits a potency of about 1100 meg/mg.

During chromatography W847-C base is converted to a hydrochloride salt by reaction with small quantities of hydrogen chloride present in the solvent. The hydrochloride, as would be expected, does not exhibit appreciable solubility in ethyl ether and is, therefore, precipitated by the addition thereof.

The free base form of W847-C may be obtained by adding dilute alkali such as .1 N NaOH to an aqueous solution of the hydrochloride to about pH 8.5-9.5 and separating the free base by filtration from the aqueous suspension.

Later fractions collected from the solumn described above when pooled and concentrated to dryness yield a white powder Antibiotic W847-C fraction. At this stage, the Antibiotic W847-C powder obtained displays a potency of about 880 mcg. mg. in the previously described cylinder cup assay.

Using the bioautograph techniques previously described (see Chemical Characterization of Antibiotic W847 Complex) and chloroformzmethanol (60:40 v./v.) as the solvent system, the Antibiotic W847-C isolate has broad spectrum antibacterial activity in vitro against the following gram-positive and gram-negative organisms: percent Bacillus subtilis ATCC 6633 Sarcina lutea ATCC 9341 Staphylococcus aureus ATCC 6538P Pseudomonas aeruginosa ATCC 8689 The remaining active fractions from the silicic acid column are combined and further resolution is achieved by Florisil (magnesium silicate) column chromatography.

Employing this technique, separation of Fraction A and Fraction B isaccomplished. Both are isolated as off-white powders by concentration of the pooled chromatographic fractions to dryness. Fractions A and B assayed at 14S meg/mg. and 71 meg/mg. respectively, in the S. lutea assay. 1

Alternatively, a mixture of Antibiotic W847-C fractions may be isolated from the ethyl acetate concentrate prepared as described hereinabove. The residue is dissolved in acetone and poured into cold water with vigorous stirring. The suspension is permitted to warm to room temperature and filtered. The precipitate comprises a mixture of Antibiotic W847-C and Antibiotic W847-C said mixture sometimes referred to as Antibiotic W847-C Complex (or Antibiotic W847-C or megalomicin-C). As precipitated, megalomicin-C is in the form of the free base. An acid addition salt of the complex may conveniently be prepared by the titration of a methanol solution of the base with the appropriate acid and by precipitating the salt by the addition of ethyl ether. When the foregoing procedure is utilized to obtain the Antibiotic W847-C Complex, Antibiotics W847-A and W847-B may be extracted from the aqueous filtrate and the extract subsequently concentrated to a residue and chromatographed on magnesium silicate as described above.

(4) Chemical Characterization of Antibiotic W847 Fractions A, B, C, and C The antibiotics of this invention are nitrogen bases; consequently, each fraction is capable of forming acid addition salts in a manner similar to that described for the Antibiotic W847-C Complex. Typical of such salts are those formed with such acids as hydrochloric, sulfuric, phosphoric, stearic, propionic, tartaric, maleic and the like. These salts display antibiotic activity similar to that of the antibiotic free base but may differ somewhat in potency and solubility characteristics.

The antibiotics of this invention are also hydroxlyated compounds; consequently, they are capable of being esterilied, preferably with a hydrocarbon carboxylic acid. Exemplary of such esters are acetates, propionates, cyclopropyl carboxylates, succinates, benzoates and the like. Such esters generally exhibit antibiotic activity similar to that of the free antibiotic usually difiering in potency and solubility characteristics.

In Table 5 below is set forth the chemical and physical properties of Antibiotic W847 Fractions A, B, C and C TABLE 5.CHEMICAL AND PHYSICAL PROPERTIES OF ANTIBIOTIC W847 FRACTIONS 7 Fraction A Fraction B Fraction C1 Fraction 0 Optical; rotation [at] D 1% in ethanol ---90 -92 1 -104 102 Melting point- 255259 dec. l25135C. dec. 243-246 C. dec. 146-150 C. dec. PKa, 9. 1 8.8 8. 8 8.6 Neutralizai ion eq 442 490 488 492 Found Calculated Found Calculated Found Calculated Found Calculated Elemental analysis:

Carbon- 60. 23 60. 25 58.90 60. 11 60. 23 59. 9s 5s. 93 60.

Hydrogen 9.28 9.19 8. 94 8.99 8.93 8.81 8.73 8.89

Nitrogen 3. 30 3. 19 2. 88 3. 05 2. 94 2. 92 2. 88 2. 87

Oxygen (by diflerence) 27. 19 27. 36 29. 18 27. 85' 27. 90 28. 30 29. 46 27. 89 Empirical formula C44HsoN2O1a 82N2 l0 4s a4N2O11 CnHsaNzOn Molecular weight 877. 10 919. 14 961. 17 975. 20

1 Monohydrate.

The column is prepared by activating the Florisil at 100 The infrared spectra of Antibiotic W847 Fractions A, C. for 16 hours; fixing in hexane and pouring the resulting B, C and C are shown in the drawings as FIGS. 1, 2, 3

slurry into a glass column. The column is charged with residual Antibiotic W847 Complex (after removal of Fractions C and C dissolved in methylene chloride and is developed progressively with one holdup volume (i.e. that volume of solvent which is held by the absorbent) of hexane, one hold-up volume of ethyl ether, one hold-up volume of ethyl acetate and then with increasing amounts of acetone in ethyl acetate. Separation is monitored by disc testing the fractions against S. aureus and E. coli followed by thin layer chromatography as previously described.

and 4 respectively. The spectra of the respective Antibiotic fractions were run in mineral oil (Nujol) and the more significant absorption peaks are set forth in Table 6 with the following designations: S=strong, M=moderate, W=weak, VS=very strong, MS=moderate to strong, brd.=broad, shp.=sharp, shd.=shoulder and s.b.-=side band.

The Antibiotic W847 Fractions A, B, C and C have characteristic nuclear magnetic resonance spectra which as shown in the drawings as FIGS. 5, 6, 7 and 8 respectively. The NMR spectra were observed on a Varian above). The results show that Antibiotic W847 Complex in activity to the other anticompares favorably biotics tested against gram-positive bacteria. In addition,

Antibiotic W847 Complex is active against Mycobacterium smegmatis at a lower concentration than erythromycin, while penicillin G and methicillin are inactive up to meg/ml.

TABLE 8.COMPARISON OF THE ANTIBACTERIAL SPEC- TRUM OF ANTIBIOTIC W847 COMPLEX WITH ERYTHRO- MYCIN, PENCILLIN G AND METHICILLIN (B) In Vivo Tests.- As was 'noted' "above; Aritibiotic W847 Complex, Antibiotic W847-C'C'omp1eX, Fractions A, B, C and 'C lexhibit a 'br oad 'ran'ge of anti-microbial activity against gram-positive and'grarn-negative microorganisms. Included in the gramp'ositiv'e group are pathogenic microorganisms includiiig species. or? the" genus Streptococcus, Staphylococcus and Diplococcus which are -'known to cause many disease manifestations. Various species "of Staphylococcus andStreptococcus are the organisms responsible for causing bovine mastitis. These species are readily controlled and treated by means of regimen of administration.

-the antibiotics disclosed herein after a r'elative'ly brief Antibiotic W847 Complex, Fractions A,- B, C .C and ,W847-C Complex are also active against gram-negative -{Salmonella Proteus and.;Pseudomonas. These organisms Erythro- Penicil- Methi- Complex myciu lin G cillin Bacillus cereus 0.2 0.05 0.12 4.8 Bacillus subtilus 0. 005 0.005 0.01 0.03 Diplococcus pneumonlae 0.2 0.05 0.12 1.8 Sarci'nalutea .0005 0.005 0.06 0.3 Staphylococcus aureus (3) 0. 005-02 0. 005-0. 05 0003-006 0.12-0.6 Staphylococcus aureus 1 0. 03-0. 6 0. 005-0. 5 .1. 2-9..4 1. 2-7. 5 Staphylococcus aureus (1 3 2. 7 50. 0 Streptococcus feacalis t .0. 005 0.0005 0.75 25.0 Streptococcus pyogenes (4) 0. 03-022 0. 05 0. 01-0. 02 0. 6 Mycobactertum amegmatls... 0.5 5.0 10 10 NOTE.Medium Al1tibi0ti0 W847 complex and erythromycin-in yeast beef broth at pH 8.0. Penicillin G and methicillin in Yeast heel broth at pH 6.8.

are responsible for manyflserious disease syndromes including urinary tracttinfections and diarrheas. Such syndromes are quite common; in domestic animals such as cattle, horses, sheep pswine, dogs and cats and may be effectively controlled and treated by means of the antibiotics described herein...i..

is greater than 5000 mg./ kg. The PD (protective dose for 50 percent of the population tested) is 150 mg./kg. by the subcutaneous route (peanut oil suspension) against a lethal infection of S. aureus in mice. Against Streptococcus pyogcnes by the. same route, the PD in mice is mg./kg.

TABLE 10 "Toxicity of antibiotic W847 fractions and con/ilpleiies and of erythromycin in mice (LDso in W847 W847 com- W847'com- W847 fraction B plex base in plex H01 in fraction base in W847-C peanut oil H2O A base i peanut oil complex Intraperitonea TABLE 11 Activity of antibiotic W847 complex and crythrouuycinalgainst lethal intraperi'toneal infections in mice (PD j sub eutaneous treatment Ant i biotic Erythromycin Antibiotic W87 base in- 'W847'com-' fraction -t: :panut 'plexHCl 'CtiHOl Peanut a oilin'HzQ' ill HjO oil HO] Staphylococcus aureus Gray 1 3 ,"5 15 6.3

Staphylococcus aureus DA 2030- Staphylococcus aureus DA 2000 Staphylococcus aureus Smith. Streptococcus pyogeues 0 DA 2 Streptococcus pyogeues DA 32 Streptococcus pyoyeues DA 31 Streptococcus pyoyeues DA 12 Streptococcus pyogeues DA 9 Diplococcus pueumouiae DA 1 Diplococcus pneumoniae DA (2 day treatment)lday Dtplococcus pncumouiae DA 151; Diplococcus pneumom'ac ATCC 10015 Staphyloccus aureus G H Streptococcus pyogez esp A 2 Diplococcus pneumoniae DA 15 1 PD loo.

13 In addition to the foregoing, Antibiotic W847 Complex administered at 17.5 mg/kg. to dogs is detectable in' the blood serum for 7 days. This is surprising in view of the fact that when a comparable'dose of erythromycin is given it does not persist throughout a 24 hour interval. 1 Acute toxicity data for Antibiotic W847 fractions and complexes are compared with erythromycin in Table 10. Tables 11,12 and 13 are set forth to illustrate the animal protection exhibited by the antibiotics of the instant invention.

TAB LE .12

Antibiotic W847 Fraction C suspended in 0.5% aqueous carboxymethyl cellulose and dispersed by ultrasonication is active in mice against S. aureus. When administered. subcutaneously against S. aureus Gray is has a PD of 62 m'g./kg. By the same route it has a PD of 180 mg./kg. against S. pyogenes C. The antibiotic is somewhat less active orally having a PD against S. wureus Gray and S. 'pyogenes C of greater than 250 mg./kg. The LD in mice by the subcutaneous route is greater than 1000 mg./kg. Antibiotic W847-C exhibits anti-malarial activity against Plasmodium berghei and when adminis- Activity of antibiotic W847 fractions and erythromyein against lethal intraperitoneal infections in mice (P1150 mg./k

Erythro- Treat- 1 a ment W847 A W847-Ci mycin route base 1 base l base 1 Streptococcus C 203 Oral 300 207 147 D0 S.C; 220 118 134 Strep a n 0 Oral 300 300 180 Do 8.0 134 180 d Streptococcus pyogenes N o. 22-.. Oral--. 180 250 D0 .C 180 200 50 Streptococcus pyoueues No. 9.. 250 250 167 250 250 180 167 75 117 40 300 250 106 70 170 75 300 250 300 160 180 250 250 250 180 250 250 200 250 250 100 r 250 160 250 250 250 150 250 250 I aerug 250 250 250 Do S.C 161.. 250 130 1 In 0.5% aqueous carboxymethylcellulose: NorE.-S.C.=subcutaneous.

:r v TABLE 13 Activity of antibiotic W847 fractions and combinations thereof against lethal intraperitoneal infections in mice I 1?Dto n mgJkg.) I

W847-B W847-C base in W847-C2 W847-C complex peanut W847-C H01 in complex HCl in v =-oi base. H10 base 1 H20 Subcutaneous administration of antibiotics;

organism: v 1

Staphylococcus aureus Gray 150 200 45 z 50 v 50 Streptococcuspyogenes C 150 10 50 50 I v uureus Smith d 86 Streptococcus pyogenes N o. 5 39 Oral administration of antibiotics; organism:

Staphylococcus aureus Gray 250 100 390 250 Streptococcus n v 0. 250 100 310 250 Staphylococcus aureus Smith Streptococcus pyogeues N o. 5

l Ultrasonicated suspension in aqueous 0.5%carboxymethyl cellulose.

Antibiotic W847-Fraction A suspended in 0.5% aqueous. carboxymethylcellulose and dispersed by. ultrasonication is active by subcutaneous administration in micev against S. aureus Gray with a PD (protective dose for 50 percent of the poulation tested) of.20 mg/kg. and against P. aerugz'nosa with a PD of 161.mg./kg. The LD in mice by the subcutaneousroute is.7000 mg-Jkg;

Antibiotic W847 Fraction .B suspended in peanut oil. is active. by subcutaneous administration inmiceagainst S. aureus with a PD of 150 mg./kg. and against P. aeruginosa, 350 mg./kg. delays death by 24 hours. The LD in mice by the, subcutaneous routeis greater than 500 mg/kg. a

tered to rodents (IOO mgJkgJ-day) 24 hours post-infection causes a significant suppression in the blood parasite.

administration. Itis, however, more advantageously used as an aqueous solution of its hydrochloride salt and when administered subcutaneously to mice has ,a PD of 45.

. .mg./kg. against S. aureus Gray and a PD of 10 mg./kg.

against S. pyogenes C. By the oral route it has a PD of 100 mg./kg. against both organisms. The LD in mice.

by intraparitoneal administration is. greater than .500

. 75 mg./kg.

Fraction C as a suspensionin aque PREPARATION OF PHYSICAL EMBODIMENTS The following examples will more fully illustrate the preparation of the antibiotics described above and constitute the best mode contemplated by applicants for carrying out their invention.

(l) FERMENTATION OF THE A MICROORGANISM EXAMPLE 1 7 Shake Flask Fermentation (A) Germination Stage.-Add a 0.5 ml. lyophilized culture of Micromonospora sp. W847 to a 300 ml. Erlenmeyer flask containing 100 ml. of the following medium Incubate the flask and its contents for 3 days at 37 C. on a rotary shaker (280 r.p.m.; 2 inch stroke).

(B) Inoculum Stage.Aseptically transfer a 25 ml. inoculum from the germination stage to a 2 liter Erlenmeyer flask containing 500 ml. of the sterile medium used above for germination. Incubate the flask andits con- At the end of this period, the potency of the produced antibiotic reaches a peak which remains substantially constant. Throughout fermentation, the pH of the fermentation mixture remains substantially in the range of 7.2 to 8.2. The packed cell volume reaches a constant value of 3.5-4.5 ml. The activity of the produced antibiotic in the whole broth gives a zone diameter of 15-25 mm. when disc tested against S. auerus or P. deruginosa.

' EXAMPLE 3 Tank Fermentation A) Preparation of Inoculum.-To four 2 liter flask each containing 500 ml. of sterile broth as described in Example 1A, add0.5 ml. of a lyophilized culture of Micromonospora sp. (W847. Place the flasks on a rotary shaker running at 280 strokes per minute (2 inch stroke) 1 and incubate at 28 C. for 72 hours.

tents for 3 days at 28 C. on a rotary shaker (280 r.p.m.;

2 inch stroke).

(C) Fermentation Stage.--Aseptically transfer 25 ml. aliquots from the inoculum stage to 2 liter Erlenmeyer flasks containing 500 ml. of the following sterile medium which has been adjusted to pH 7.8 with dilute aqueous sodium hydroxide prior to sterilization.

Gm. Sucrose 30.0 Sodium nitrate 3.0 Dipotassium phosphate 1.0 Magnesium sulfate 0.5 Ferrous sulfate 0.01 Peptone 8.0

Tap water, 1000.0 m1.

Incubate the flasks and their contents for 4 days at 28 C. on a rotary shaker (280 r.p.m.; 2 inch stroke). Pool the contents of the flasks and adjust. analiquot of the whole broth to pH 9.5 and extract with ethyl acetate (approximately 2 volumes per volume of broth). Separate the solvent phase and concentrate 100 fold under vacuum. Test the concentrate for activity by the agar disc diffusion technique using S. aureus (ATCC 65381) as the test ortraction (about 3 7 liters).

ganism. The concentrate will give a zone 'of inhibition of approximately 20-35 mm. 1

EXAMPLE 2 Tank Fermentation (A) Germination Stage-Carry out the germination stage and the inoculum stage in the manner described.

in Example 1.v

(B) Fermentation Stage.Aseptically transfer 500 ml.

ofinoculum to a 14 liter fermentor containing 10 liters of sterile medium as described in Example 1 to which has Temperature: 31 C. Sterile air input: 4 liters/minute Agitation: 250 r.p.m.

Anti-foam (Dow Corning B) Add as needed (B) Fermentation Stage.-To each of four fermentors, add 10 liters of the following sterile production medium adjusted to pH 7.15 to 7 .25 prior to sterilization:

Trypticase 170.0 Sodium Chloride 50.0 Dipotassium Phosphate 25.0 Dextrose 25.0 Czapek-Dox Broth 350.0

G.E.- Antifoam (General Electric Company,

Schenectady, N.Y.). Tap water, q.s., 10.0 liters Inoculate each fermentor with 500 ml. of the 72 hour seed culture prepared above. Bring the temperature of the ferentation medium to 31 C. and agitate at 500'r.p.m. while introducing air flow through the medium at the rate of 0.5 liters of air/liter of broth/minute. Increase the agitation rate to 600 r.p.m. after 24 hours and to 700 r.p.m. after 48 hours. Terminate the fermentationat the end of 69 hours. Pool the broths from the four fermentors for ex (2) EXTRACTION OF ANTIBIOTIC W847 COMPLEX EXAMPLE 4 Extraction of Antibiotic W847 Complex from Laboratory Fermentation (Chromatographic Technique) Adjust 60 liters of whole broth prepared according to' the procedure described in- Example 2 to pH 9.5 with dilute aqueous sodium hydroxide. Extract with 2 volumes of ethyl acetate for each volume of broth. Separate the solvent phase and concentrateunder vacuum to obtain an oily residue (approximately 30.0 g.). The biological activity (1/ 20 dilution) of this oily residue will givea zone of inhibition diameter of about 20-30 mm. against S. aureus and about 15-25 mm. against P. aer uginosa. Purify the oily residue by column chromatography accordingto the following technique:

Prepare a column using 1000 g. 'of LH20 Sephadex (Pharmacia Fin Chemical, Inc.) suspended-in 95 percent aqueous ethanol. Transfer the oily residue to the column and elute with percent ethanol at a flow rate of 200 ml./hr. Collect 50 ml. fractions. Combine fractions according to their antibacterial activity (determined by agar disc testing against S. aureus. ATCC '6538P). Concentrate fractions having peak activity to dryness. Dissolve the solid residue in a small amount of acetone and pour into an excess volume of petroleum ether (B.P. 30-60 C.). Transfer the mixture to a Dry Ice-acetone bath (approximately '50 C.) and allow'to stand 20 minutes. Allow the mixture to return to room temperature and separate the mother liquor from the oily residue by'decanting. Concentrate the mother liquor todryness to obtain purified Antibiotic W847 Complex.

Antibiotic W847 Complex so produced will havea potency of about 225 to 275 meg/mg. when assayed according to the cylinder cup technique described above.

17 EXAMPLE Extraction of Antibiotic W847 Complex From Tank Fermentation (Solvent Extraction Technique) Adjust the pooled broth (about 37 liters) obtained as described in Example 3 to pH 9.5 with 50 percent aqueous sodium hydroxide. Extract with two volumes of ethyl acetate and evaporate the extract to a volume of 2,150 ml. Isolate the Antibiotic W847 Complex by one of the following techniques:

' -(A)Hydrochloric Acid Extraction -Extract a 500 ml. aliquot of the ethyl acetate concentrate twice with 250 ml. portions of 0.5 percent (0.14 N) hydrochloric acid. Make the combined acid extracts slightly alkaline by the addition of 5 percent aqueous sodium hydroxide (about pH 8.5 to 9.0) and extract twice with 250 ml. portions of ethyl acetate. Combine the ethyl acetate extracts and concentrate under reduced pressure to dryness. The complex so produced will have a potency of about 525 mcg./ mg. when assayed by the previously described cylinder cup technique (B) Sulfuric Acid Extraction.Repeat the extraction tained as a white powder by concentrating the selected column fractions to dryness. At this stage, this material exhibits a potency of about 900 meg/mg. in the cylinder cup assay previously described. The remaining active material is eluted from the column, pooled and concentrated to a residue.

' EXAMPLE 7 Isolation of Antibiotic W847 Fraction A and Fraction B Activate 500 g. of Florisil at 100 C. for 16 hours; fix in hexane and slurry. into a glass column (105 cm. long; 3.8 cm. diameter; hold-up volume 700 ml.). Charge the column with 2.8 g. of the residue from Example 6, dissolved in 10 ml. of methylene chloride. Elute the'column progressively with 700 ml. of hexane, 700 mljof ether, 700 ml. of ethyl acetate and increasing amounts of acetone in ethyl acetate (flow rate 200 ml./hr.). Collect 100 ml. fractions. The column is monitored as previ ously described by testing fractions against S. aureus and E. coli followed by thin layer chromatography. Combin the fractions as indicated in Table 14 below.

TABLE 14.RESULTS OF COLUMN SEPARATIONS Thin layer pattern R1 chlorotorrmmethanol Potency Fraction Column cut Weight (60/40) (meg/mg.)

Silicic acid column:

470z,HOl 28-48 1.2 g 0. 65 900-1, 000 W847Cz- 56-58 625 mg 0. 52 850-1, 000 Florisil colum:

W347B 164-227 (30% acetone in ethyl acetate) 540 mg..-. 0.38 125-175 ws67A 370-397 (80% acetone in ethyl acetate) 283 mg 0. 19 50-100 described in Example 5A substltuting 0.1 N sulfuric acid EXAMPLE 8 for the hydrochloric acid. The complex so produced will have a potency of about 550 mcg./ mg. in the cylinder cup assay.

(3) RESOLUTION OF ANTIBIOTIC W847 COMPLEX EXAMPLE 6 7 Isolation of Antibiotic W847 Fraction C Prepare a column (105 cm. long, 7.6 cm. diameter) using 2000 g. of silicic acid (Analytical Reagent, Mallinckrodt) 'by' packing small segments into a compact bed. Dissolve 13.0 g. of Antibiotic W847 Complex as prepared in Example 4 in acetone mixed with silicic acid and evaporate the acetone under vacuum. Add the dry mixture to the top of the column. Elute the column with a mixture of chloroform (60 parts by volume) and methanol (40 parts low, Antibiotic W847 Fraction C Hydrochloride is isolated as a White powder by dissolving the combined residues in one volume of methanol and precipitating with 10 volumes of ethyl ether. This material at this stage has a potency of about 1000 meg/mg. in'the cylinder cup assay previously described.

Isolation of Antibiotic W847 Fraction C Material By combining later fractions as indicated in Table 14 below, an Antibiotic W847 Fraction C isolate is ob- Antibiotic W847 Complex Hydrochloride Salt Suspend about 140 mg. of Antibiotic W847 Complex, as isolated in Example 5 above, in about 50 ml. of water. Adjust the pH of the suspension to about 6.5 to 7.0 by the addition of dilute (1 N) hydrochloric acid and stir .vigorously. Remove the insolubles, if any remain, by filtration and lyophilize the filtrate to obtain an Antibiotic W847 hydrochloride weighing approximately mgs. and assaying about 550 meg/mg.

By the utilization of substantially the procedure of this example on either the Antibiotic W847 Complex or the various fractions thereof and by substituting equivalent quantities of such acids as sulfuric, phosphoric and the like, their respective acid addition salts may also be prepared. Applicants consider all such salts to be the full equivalent of the antibiotics described herein and to fall within the scope of their invention.

Table 15 below sets forth the activity of the hydrochloride, sulfate and phosphate salts of Antibiotic W847 Complex against a variety of gram-positive and gramnegative organisms.

TABLE 15.ANTIBACTERIAL SPECTRUM OF ACID ADDITION WATER SOLUBLE SALTS OF AN TIBIOTI W847 COMPLEX C Minimum inhibitory concentration (meg/ml.)

Hydro: Phoschloride Sulfate phate Microorganism salt salt salt Bacillus subtilis ATCC 6633 0. 01 0.01 0. 0|. Staphylococcus aureus ATCC 6538P 0. 1 0. 01 0. 1 Stapltylococcus aureus ATCC 11163 0. 1 0. 1 0. 1 Sarcma Zutea ATCC 9341 0. 001 0.001 0. 001 Streptococcus faecalis ATOC 1054 0. 1 0. 1 0. 1 Eschenchta coli ATCC 10536- 2. 8 2. 8 2. 8 K lebsiella pueumoniae ATCC 10031 1. 4 1. 4 2. 8 Proteus vulgaris DA 121 5. 6 11. 1 11. 1 Pseudomtmas aerugiuosa ATCC 8689-.-.- 1. 4 1. 4 1. 4 Salmonella schottmuelleri DA 10 1. 4 2. 8 2. 8

NorE.-Medium=yeast beet broth pH 8.0. Salts made from antibiotic W847 complex with a potency of 232 meg/mg.

"EXAMPLE9 f'; Isolationjof Antibiotic W847-C Complex Dissolve 542 grams of 'W847 Complex (see ExampleS) in 5.4 liters of ;aceto ne and treat 'theresulting solution with 81 grams of decolorizing carbon for about 30 min utes at about room temperature. Remove the decolorizing carbon by filtration and concentratethe filtrate in vacuo to approximately 2.0"liters. Prepare a slurry of about 80 liters ofii'cexa'nd waterand with vigorous stirring add the acetone" solution. Allow the temperature of the resulting suspension to rise to about 25 C. with stirring and collectthe product by'filtration. Wash the solids with a small quantity of water and dry at about 50 C. in vacuo to yield about 212 grams of Antibiotic W847-C complex assaying about 1075 meg/mg.

-Additional product (about 85 grams) may be obtained b'yrconcentration of the mother liquor, however, this product mayi'be contaminated with Fractions A and B.

Antibiotic-W847-C Complex as prepared above may advantageously be used in the preparation of acid addi-. tion salts as shown below.

EXAMPLE Preparationofa Tartrate Salt of An Antibiotic W847-C Complex To a solid composition comprising 2.0 g. of a complex of Antibiotic W847-C and Antibiotic W847-C obtained in Example 9 above,'add 30 ml. of diethyl ether with stirring. Add to the resulting solution 0.5g. of tartaric acid dissolved in 2.5 ml. of ethanol. Stir the mixture for about minutes, then filter the precipitated salt. Wash the precipitate with diethyl ether and dry the product to obtain about 2.0 g. of tartrate salt whose bioassay is about 575 mcg./mg. and whose rotation is (-57) concentration 1% in ethanol.

EXAMPLE 11 Antibiotic W847-C Complex Propionate wa 110g. of ws47-c ,Complex in 55 ml; or pyridine and add 5.5 mlof acetic'anhydride. Stir the reactionmixture at room temperature for about 18 hours,

then pour itinto 2.0 1. of ice water containing 50 ml. of 14% ammonium hydroxide with vigorous stirring. Filter the resulting precipitate, wash 'with water and dry to yield the desired product melting 237-24 1 C. and having a rotation of about 86 0:1 in ethanol.

Alternatively, the precipitated product may be extracted with ethyl acetate, the extract washed with water and evaporated in vacuo to a residue. The residue is subsequently dissolved in acetone, treated with decolorizing carbon and precipitated by the addition of anequal yolume of water to yield the product of this example.

EXAMPLE l3 Antibiotic W847-C Diacetate h v V Dissolve 10.0 g. of Antibiotic W847-C 'in'50 ml. of pyridine; add 5.0 ml. of acetic anhydride andfkeepthe mixture at room temperature for about 18 'hours'. Pour the solution into 2 liters of ice water and add 50 ml. of 14% ammonium hydroxide. Filter the resulting suspen- 20 sion, wash the precipitate with water and dry. Crystallize the precipitate from aqueous acetone, treating with decolorizing' carbon if needed, to obtain the title product, m.p. 240 -'243 C.,'[a] =-86 (C- '1, ethanol).

EXAMPLE 14 1 Antibiotic W847-C Dipropionate Dissolve 10.0 g. of Antibiotic W847-C in 50 m'l'pof'pyridine; add 5.0 ml. of propionic anhydride and hold the resulting solution at room temperature for about 18 hours. Pour the solution into 2 liters of ice water and add 50 ml. of 14% ammonium hydroxide. Extract the product with methylene chloride and wash'the extract with'water. Concentrate the extract to a residue and crystallize the residue from aqueous acetone to obtain the product of this ex: ample, m.p. 17477 C., [a] -'84. (C=1,

ethanol). 7 v

EXAMPLE 15 Antibiotic W847-B Diacetate Dissolve 5.0 g. of Antibiotic W847-B in 25 ml. of pyridine; add 2.5 ml. of acettic anhydride and hold the resulting solution at room temperature for about 18 hours. Concentrate the pyridine solution in vacuo below about 40 C.'Dissolve the residue in acetone and dilute the acetone solution with 5% ammonium hydroxide to yield the title product as a crystalline suspension. Filter the suspension and wash. If desired, the product may be re"- crystallized from aqueous acetone to yield Antibiotic W847-B Diacetate, m.p. 198210 C., [a] =79 (C: 1, ethanol).

' EXAMPLE l6 Antibiotic W847-A Tripropionate Dissolve 10.0 g. of Antibiotic W847-A in 200 ml. of pyridine; add 20.0 ml. of propionic anhydride and hold the mixture at room temperature 'for about 18 hours. By the isolation method of Example 15, there is obtained the product of this example, m.p. 177-179 C.,

Suspend 250 mg. of Antibiotic W847-A and 7150 mg. of sodium bicarbonate in 4 ml. ofacetone. Add mg. of benzoyl chloride and stir the mixture at room temperature for about 4 hours and filter the mixture. Concentrate the .filtrate under reduced pressure to a'residue. Dissolve the residue in about 5- ml. of acetone and dilute slowly with 5% ammonium hydroxide. Filter the'uresulting precipitate, wash with water and dry to obtain the product of this example, mp. 222 -225 C.

H EXAMPLE... 19 Antibiotic ws47 A Tribenzoatei Dissolve 250 mg. of Antibiotic W84f7-A in 5 -ml. of pyridine and add 0.14 ml. of benzoyl chloride with stirring. Keep the solution at room temperature overnight, then concentrate under reduced-pressure-to about 1 ml.

and dilute with about m1. of acetone. Add 5% ammonium hydroxide until the product precipitates. Filter the suspension, wash the precipitate with water and dry to obtain the product of this example, m.p. 144149 C.

EXAMPLE Antibiotic W847-C Dibenzoate Esterify Antibiotic W847-C with benzoyl chloride according to the procedure of Example 18. Isolate the product as described and obtain the title product, m.p. 166-169 C.

EXAMPLE 21 Antibiotic W847-A Triacetate To a esolution of 10.0 g. of megalomicin-A (from Example 7) in 200 ml. of pyridine add 20.0 ml. of acetic anhydride and keep the reaction mixture at room temperature for 18 hours. Work up the reaction mixture according to the procedure described in Example 13. Crystallize the product from aqueous acetone to obtain the compound of this example, m.p. 199 202 C., [a] '=86 (C=1, ethanol).

By substituting an equivalent quantity of isovaleric anhydride for the acetic anhydride in the preceding example and by following the procedure of said example, Antibiotic W847-A tri-isovalerate is obtained.

Similarly, by substituting an equivalent quantity of stearoyl chloride for the acetic anhydride in Example 21 and by following the procedure of said example, Antibiotic W847-A tristearate is obtained.

EXAMPLE 22 Antibiotic W847-A Triacetate Lauryl Sulfate Dissolve 250 mg. of Antibiotic W847A triacetate, from the preceding example, in 3 ml. of acetone and add a solution of sodium lauryl sulfate in 3 ml. of water. Add with stirring a mixture of 0.03 ml. of acetic acid and 0.2 m1. of water. Dilute the mixture to a volume of ml. with water and refrigerate overnight. Decant the supernatent and dissolve the waxy residue in acetone. Pour the acetone solution into ether, filter the resulting suspension and dry the precipitate at room temperature to obtain the compound of this example, m.p. 13540 C.

EXAMPLE 23 Megalomicin-A Hemisuccinate To 1.0 g. of megalomicin-A in 20 ml. of acetone add 228 mg. of succinic anhydride. Heat the mixture to boiling and remove any insoluble material by filtration. Heat the filtrate briefly at reflux, then allow to stand at room temperature for 18 hours. Dissolve the oily precipitate by warming the mixture, concentrate to one half volume, add a small amount of methanol and pour slowly with agitation into 200 ml. of ether. Filter the resulting precipitate, wash with ether and dry to obtain a product which TLC and bioautography shows to consist of two active components. Separate by chromatography on silica gel to obtain megalomicin-A di-hemisuccinate, m.p. 163 166 C. and megalomicin-A tri-hemisuccinate, m.p. 165l67 C.

In view of the foregoing examples, it is apparent that the antibiotics of this invention will form esters with a host of other acyl residues and applicants consider all such esters to be the full equivalent of those whose preparation is described herein.

The following examples are directed to some of the pharmaceutical dosage forms wherein the antibiotics may 'be used. The formulations, excluding the topicals, are designed to permit the administration of from about 5 to about 50 mg. of antibiotic per kilogram of body weight per day. It is contemplated that the medicaments be given in doses up to 8 times in a 24 hour period.

The topical formulations are generally applied to the infected area from 2 to 4 times daily.

22 In the utilization of the formulations set forth below the dosage of antibiotic given and the frequency of administration depends in large part of the stage and severity of the infection, the type of infection and the individual characteristics of the animal species being treated.

EXAMPLE 24 Capsule Mg. Antibiotic W847 Complex 250.00 Lactose 248.75 Magnesium stearate 1.25

Procedure 1. Blend the Antibiotic W847 Complex and the lactose. 2. Add the magnesium stearate and mix. 3. Fill capsules.

EXAMPLE 25 Oral Suspension (to give a close of 125 mg. per 5 ml.)

Gm. Antibiotic W847-A 25.0 Magnesium aluminum silicate 9.5 Sodium carboxymethylcellulose, U.S.P. 2.5 Sodium Citrate, U.S.P. 25.0 Flavor Q.s. Color Q.s. Methylparaben, U.S.P. 0.9 Propylparaben, U.S.P. 0.2 Polysorbate 80, U.S.P. 1.0 Sorbitol Solution, U.S.P. 500.0

Water, q.s., 1000.0 ml.

Procedure 1. Heat 200 ml. of water to boiling, and dissolve in it one half of the parabens. Cool to about 70 C., then mix in the Polysorbate 80. Sprinkle in the silicate, stirring until a uniform smooth suspension results.

2. Heat an additional 200 ml. of water to boiling, and dissolved in it the remainder of the parabens. Disperse the CMC in this until a smooth gel results. Mix in the sorbitol Solution. Then dissolve the sodium citrate 3. Add 2 to 1 slowly, with constant stirring. Cool the mixture to 25 C. Add the Antibiotic W847A, flavor Procedure 1. Heat the stearic acid, sorbitan monostearate, sorbitan monooleate, and polyoxyethylene sorbitan monolaurate to 65 C.

2. Heat about of the water to 7 0 C.

3. Add the water to Step 1 and mix to form the cream base.

4. Slurry the Antibiotic W847B with about 10% of the water and pass through a colloid mill.

5. Add the milled slurry to the molten base and mix. Allow to cool.

EXAMPLE 27 Topical Ointment Gm. Antibiotic W847-C 1O Petrolatum 990 Procedure 1. Melt the petrolatum.

2. Slurry the Antibiotic W847C with about 10% of the petrolatum and pass through a colloid mill.

3. Mix the milled slurry with the remainder of the molten petrolatum. Allow to cool.

EXAMPLE 28 Parenteral Suspension Mg. Antibiotic W847-C 250 Sodium carboxymethylcellulose 3 Polysor-bate 80 USP 1 Methylparaben 3.6 Propylparaben 0.4

Water, q.s., 2.0 ml.

Procedure 110-- CH; H1) H3 H3C- -o on. o l 0 HO H3 O y WW 2115 0 CH3 CH: 0 R1 0 l CH3 including the non-toxic pharmaceutically acceptable acid addition salts thereof, wherein R and R respectively, are selected from the group consisting of (R =H, R =H) in megalomicin A; (R =H, R =acetyl) in megalomicin B; (R =acety1, R =acetyl) in megalomicin C and (R =acetyl, R =propionyl) in megalomicin C 2. A composition of matter as defined in claim 1, said composition being a non-toxic pharmaceutically acceptable hydrocarbon carboxylic acid ester of megalomicin A and the non-toxic pharmaceutically acceptable acid addition salts of said ester.

3. A composition of matter as defined in claim 1, said composition being a non-toxic pharmaceutically acceptable hydrocarbon canboxylic acid ester of megalomicin B and the non-toxic pharmaceutically acceptable acid addition salts of said esters.

4. A composition of matter as defined in claim 1, said composition being a non-toxic pharmaceutically acceptable hydrocarbon carboxylic acid ester of megalomicin C and the non-toxic pharmaceutically acceptable acid addition salts of said ester.

5. A composition of matter as defined in claim 1, said composition being a non-toxic pharmaceutically accept-. able hydrocarbon carboxylic acid ester of megalomicin C and the non-toxic pharmaceutically acceptable acid addition salts of said ester.

6. A composition of matter as defined in claim 1, said composition being a non-toxic pharmaceutically acceptable hydrocarbon carboxylic acid ester of megalomicin C complex and the non-toxic pharmaceutically acceptable acid addition salts of said ester, said complex consisting of megalomicin C and megalomicin C 7. A compound of claim 2, said compound being megalomicin A dipropionate.

8. A compound of claim 2, said compound being megalomicin A tripropionate.

9. A compound of claim 2 said compound being megalomicin A dibenzoate.

10. A compound of claim 2 said compound being megalomicin A tribenzoate.

11. A compound of claim 2, said magalomicin A triacetate.

12. A compound of claim 2, said megalomicin A triacetate lauryl sulfate.

13. A compound of claim 2, said megalomicin A dihemisuccinate.

14. A compound of claim 2, megalomicin A 'trihemisuccinate.

15. A compound of claim 3, megalomicin B diacetate.

16. A compound of claim 4, megalomicin C diacetate.

17. A compound of claim 4, megalomicin C dibenzoate.

18. A compound of claim 5, megalomicin C dipropionate.

19. A compound of claim 6, said megalomicin C complex propionate.

20. A compound of claim 6, said megalomicin C complex diacetate.

compound being compound being compound being said compound being said compound being said compound being said compound being said compound being compound being compound being References Cited UNITED STATES PATENTS 3,634,393 1/1972 Motola 260-210 AB 3,669,953 6/1972 Mallams 260-210 AB JOHNNIE R. BROWN, Primary Examiner US. Cl. X.R. 31

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION PATENT NO. 3,819 ,611 DATED June 25 197 INVENTOR S Marvin J. Weinstein, Gerald H. Wagman, George M.

Luedemann and Joseph A, Marc? It IS cerhfled that error appears In the above-ldentlfled paten are hereby corrected as shown below:

uez and that sand Letters Patent Column 1, line 25 "indivdual" should read --individual--. Column line 6 "800 meg/ml" should read --800 mcg/ml--. Column 4 line 4-5 "Dry Ice" should read --dry ice--.

Column 6, Table t "Crythromycin" should read --erythromycin--. Column 6, line 25 "Antio'botic" should read --Antibiotic--. Column 6, line & "ethyl acetate methanol and acetone" should.

read --ethyl acetate, methanol ethanol and acetone--. Column 6 line 6h "hereinafter as" should read --hereafter as--. Column 7, line 28 "from the solumn described above" should read --from the column described above--. Column 9 Table 6 "(1)" should read --(instrument artifact)--. Column 9 Table 6 under Fraction C "(1)" should read --(instrument artifact)--. Column 10, Table 7 under Fraction 3 "9.5" should read --O.5--. Column 10, Table 7 under Fraction B "0.05" should read --0.005-- Column 10, Table 7 under C-complex "O. 0075" should read --0.000T-. Column 11, Table 8 under complex "0.05-022" should read --0.05-0.22--. Column 15 line 7 "erythromycin in Table 10" should read --erythromycin in Table 10 below--. Column Table 12 "8.0." should read --S.C. Column 16, line 15 "Micromonospora sp (WB TT' should read --Micromonospora sp w8 l-T--. Column 17, Table 1h "W86'TA" should read --W8 LTB--. Column 19 line T5 "hyddoxide" should read --hydroxide--. Column 22 line +5 "dissolved in it" should read --dissolve in it--. Column 22 line "add sufficiently" should read --add sufficient--.

Signed and Scaled this ninth Day of December1975 [SEAL] A ttes t:

Arresting Officer UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION PATENT NO. 3,819 ,611 DATED June 25 197 INVENTOR S Marvin J. Weinstein, Gerald H. Wagman, George M.

Luedemann and Joseph A, Marc? It IS cerhfled that error appears In the above-ldentlfled paten are hereby corrected as shown below:

uez and that sand Letters Patent Column 1, line 25 "indivdual" should read --individual--. Column line 6 "800 meg/ml" should read --800 mcg/ml--. Column 4 line 4-5 "Dry Ice" should read --dry ice--.

Column 6, Table t "Crythromycin" should read --erythromycin--. Column 6, line 25 "Antio'botic" should read --Antibiotic--. Column 6, line & "ethyl acetate methanol and acetone" should.

read --ethyl acetate, methanol ethanol and acetone--. Column 6 line 6h "hereinafter as" should read --hereafter as--. Column 7, line 28 "from the solumn described above" should read --from the column described above--. Column 9 Table 6 "(1)" should read --(instrument artifact)--. Column 9 Table 6 under Fraction C "(1)" should read --(instrument artifact)--. Column 10, Table 7 under Fraction 3 "9.5" should read --O.5--. Column 10, Table 7 under Fraction B "0.05" should read --0.005-- Column 10, Table 7 under C-complex "O. 0075" should read --0.000T-. Column 11, Table 8 under complex "0.05-022" should read --0.05-0.22--. Column 15 line 7 "erythromycin in Table 10" should read --erythromycin in Table 10 below--. Column Table 12 "8.0." should read --S.C. Column 16, line 15 "Micromonospora sp (WB TT' should read --Micromonospora sp w8 l-T--. Column 17, Table 1h "W86'TA" should read --W8 LTB--. Column 19 line T5 "hyddoxide" should read --hydroxide--. Column 22 line +5 "dissolved in it" should read --dissolve in it--. Column 22 line "add sufficiently" should read --add sufficient--.

Signed and Scaled this ninth Day of December1975 [SEAL] A ttes t:

Arresting Officer 

